Realization of a soft-MI electrospun scaffold for tissue engineering applications

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PP82

Layer-by-layer functionalisation of polymeric

blends to favor stent endothelialisation

I Carmagnola

1

, V Chiono

1

, S Cabodi

2

, F Logrand

2

and G Ciardelli

1

_{Department of Mechanical and Aerospace Engineering,}

Politecnico di Torino, Torino, Italy;

2

Department of Molecular

Biotechnology and Health Science, Molecular Biotechnology

Center, University of Turin, Torino, Italy

Introduction: Speciﬁc ligand immobilisation can enhance endothelial cells (ECs) adhesion on stent surface, avoiding migration of smooth muscle cells (SMCs) [1]. Wei et al. [2] demonstrated that the REDV peptide promote the EC growth respect to SMCs. In this work, poly-meric substrates were coated through layer-by-layer technique to func-tionalize the_{ﬁnal layer with the CRETTAWAC sequence able to favor} ECs and inhibit platelet attachment [3].

Materials and methods: Aminolysed poly(L-lactide)/poly(DL-lac-tide-co-glycolide) (PLLA/PLGA)_{ﬁlms were dipped into alternate} aque-ous solutions (0.5% w/v, pH: 4.5) of poly(sodium 4-styrene sulfonate) (PSS) and poly(diallyldimethylammonium chloride) (PDDA) for 15 minutes each, with intermediate washing steps in bidistilled water (pH: 4.5) for 5 minutes. Heparin (HE) was deposited as the last layer of the coating. Surface composition was evaluated through XPS analysis and via colorimetric method. The CRETTAWAC effect on re-endothelial-ization was evaluated using human umbilical vein endothelial cells (HUVECs). Cells were seeded onto three different surface: CRETTA-WAC, bovine serum albumin (BSA) andﬁbronectin (FN).

Results: The deposition of multilayer coating was conﬁrmed by XPS analysis.

Table 1. Atomic percentage of chemical elements of functionalised sam-ples.

C1s O1s N1s S2p

Aminolyzed PLLA/PLGA 64.0 35.3 0.7 –

PLLA/PLGA+ 14L 71.2 26.0 1.3 1.5

PLLA/PLGA+ 15L 70.2 25.8 2.2 1.8

After the deposition of 14 and 15 polyelectrolyte layers the percent-age of N and S was higher respect to aminolysed PLLA/PLGA due to the deposition of PDDA, PSS and HE (Table 1). In vitro tests show that after 2 hours the number of adhered cells on CRETTAWAC functiona-lised substrate were less respect to FN. After 4 hours HUVECs seeded on CRETTAWAC showed a spread shape.

Discussion and conclusions: Through the LbL technique allows to obtain functional coatings without changing the bulk properties. CRETTAWAC sequence was shown to bind the a5b1 integrin that is present on the surface of ECs but lacking on platelets. LBL could be exploited for sur-face functionalisation with KKKKKKSGSSGKCRRETAWAC, able to elec-trostatically interact with HE.

Acknowledgments: NANOSTENT (Regional Project, Regione Pie-monte).

References

1. Smith E.J., Rothman M.T., J. Interv. Cardiol. 16, 475, 2003. 2.Wei Y., Ji L-L., Xiao Q-k., Lin J-p., Xu K-f., Ren J. Ji, Biomaterials 34, 2588, 2013.

3. Meyers S.R., Kenan D.J., Khoo X., Grinstaff M.W., Biomacomolecules 12, 533, 2011.

PP83

Realization of a soft-MI electrospun scaffold for

tissue engineering applications

G Criscenti

1

, G Cerulli

2

, D Saris

1

, C Van Blitterswijk

1

, G Vozzi

3

,

H Fernandes

1

_{and L Moroni}

1

_{Department of Tissue Regeneration, MIRA Institute for}

Biomedical Technology and Technical Medicine, Faculty of Science

and Technology, University of Twente, Enschede, The Netherlands;

2

_{Istituto di Ricerca Traslazionale per l’Apparato Locomotore –}

Nicola Cerulli

– LPMRI, University of Perugia, Arezzo, Italy;

3

_{Research center}

_{“E. Piaggio”, Faculty of Engineering, University}

of Pisa, Pisa, Italy

Introduction: The fabrication of bioactive scaffolds able to mimic the in vivo cellular microenvironment represents a challenge for tissue engineering (TE). In the present work, we propose the combination of electrospinning (ESP) and soft-molecular imprinting (soft-MI) [1] to create a soft-molecular imprintedﬁbrous scaffold for TE applica-tions. By combining these techniques, we fabricated poly-L-lactide acid (PLLA), PolyActive (PA) and poly lactic-co-glycolic acid (PLGA) based scaffolds functionalized using different protein arranged onto the surface.

Materials and methods: A functionalized PDMS mold was used as a tar-get for the ESP jet. The PDMS mold was prepared using a commercial product (Sylgard 184 kit, Dow Corning, MI) with a 10:1 (w/w) ratio. The mixture was casted onto a silica wafer comprising different cus-tom-made geometries, degassed, and _{ﬁnally cured in an oven for} 4 hours at 70_{°C. To reduce the hydrophobicity of its surface, the PDMS} mold was treated with Piranha Solution (3:1). After the derivatization of its surface with a 1% solution of (3-Aminopropyl)-trimethoxysilane in Toluene, the PDMS mold was activated with an EDC-NHS solution. Finally, the mold was immersed in a protein solution for 24 hours. The proteins used as templates were FITC-albumin and TRITC-lectin. Three different polymer solutions were prepared: PLGA 4% (w/v) in 1,1,1,3,3,3-hexaﬂuoro-2-propanol (HFIP), PLDLA 5% (w/v) in HFIP and PA300 20% (w/v) in CHCl3/HFIP 70:30 (v/v). The functionalized

PDMS mold was positioned under the ESP jet and the ESPfibers fabri-cated at aflow rate of 1 mL/h and a voltage of 20 kV. The imprinted scaffolds were tested for their ability to rebind the template protein and observed with afluorescence microscope. To evaluate the selectivity for a particular template molecule, the imprinted scaffolds were immersed in a solution of both FITC-albumin and TRITC-lectin. The obtained scaffolds were analyzed by_{fluorescence microscopy to ensure that no} weakly bound protein on the PDMS was transferred to them.

Figure 1. Microscopy images of adhered HUVECs on different func-tionalised surface after 4 hours.

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Results: The imprinted scaffolds demonstrated affinity and selectivity for their template molecule over the otherfluorescent protein competi-tor. Specifically, the FITC-albumin imprinted scaffolds showed protein absorption in a solution of FITC-albumin (Fig. 1a) while no protein adsorption was seen in a solution of TRITC-lectin. Similar results were obtained with the TRITC-lectin imprinted scaffolds (Fig. 1b).

Figure 1. (a) FITC-albumin imprinted PLGA scaffold; (b) TRITC-lectin imprinted PLGA scaffold.

Discussion and conclusions: The integration of ESP and soft-MI repre-sents a promising technique for the realization of scaffolds for TE appli-cations, because it allow us to mimic the biological environments through the selective bindings of speciﬁc endogenous proteins. Reference

1. Vozzi G. et al., Biotechnology and bioengineering, 2010. 106(5), 804_–17.

PP84

Electrospun microyarns for vascularization

in tissue engineering

K Rietzler

1

_{, K Feher}

1

_{, S Weinandy}

2

_{, M Kruse}

1

_{, P Schneider}

3

_,

T Gries

1

and S Jockenhoevel

2

1

_{Institut fuer Textiltechnik an der RWTH Aachen University,}

RWTH Aachen University, Aachen, Germany;

2

_{AME-Helmholtz}

Institute for Biomedical Engineering, RWTH Aachen University,

Aachen, Germany;

3

Department for Textile Technology- Technical

Textiles, HS Albstadt- Sigmaringen, Albstadt, Germany

Introduction: As an interdisciplinary_{field Tissue Engineering is aiming} at the reproducibility of damaged tissue or organs. Although there are constantly achieved successes in the design of tissue engineered con-structions, there is still the dif_{ficulty to provide those with a sufficient} vascularization. Successful applications in Tissue Engineering involve thin avascular tissue like cartilage and skin without a vasculature.

Aim of the present research work is the production of electrospun mi-croyarns to support angiogenesis in_{ﬁbrin gel scaffolds.}

Materials and methods: Microyarns with different morphological prop-erties (polymer concentration, polymer blend, diameter, torsion angle) were produced by electrospinning[1]_{. Polylactide (PLA) and}

polyethyl-ene glycol (PEG) were used as polymers. The electrospun microyarns were UV- sterilized for 30 minutes under the laminarﬂow hood. After sterilization the microyarns were seeded with 1.49 106human umbili-cal vein endothelial cells (HUVECs) and incubated (37°C, 5%CO2) in

24-well plates for 24 hours. Subsequently the microyarns were stained with 40,6-Diamidin-2-phenylindol (DAPI) and analyzed under the ﬂuo-rescence microsope[2]_{. Those of the microyarns that showed the best}

proliferations were used for further experiments. To proof their capabil-ity of developing capillary-like structures, the microyarns were culti-vated in 3D. The micoyarns were_{first seeded in 24 well plates with} HUVECs; then embedded in_{fibrin gel containing both HUVECs and} human dermal foreskin_{fibroblasts (HDFFs). After 14 days of} co-culti-vation the_{fibrin gels were fixed and immunostained (CD31).} Verifica-tion and quanti_{fication of the capillary-like structures were} implemented by two-photon laser scanning microscopy (TPLSM) and ImagePro Analyzer software. All experiments were replicated three times with different donors.

Results: After 14 days of co-cultivation, it could be observed that there is formation of capillary-like structures within the HUVEC/HDFF co-cul-ture system. There were also signiﬁcant distinctions in formation between the different morphological properties of the microyarns.

Figure 1. Cell adhesion on PLA/PEG microyarn (l), SEM image of PLA/ PEG microyarn (r).

Discussion and conclusions: The present work shows, that microyarns can be applied as textile scaffolds for vascularization. Depending on their morphological property, they are also capable to inﬂuence the ori-entation of cell adhesion.

References

1. Dalton P., Klee, D. et al., Polymer 46:3, 611-614. 2005.

2. Sorrell J, Baber M, et al., Cells Tissues Organs.186:157-68. 2007.

PP85

Fabrication of a functionally layered membrane

designed for periodontal tissue regeneration

P Gentile, JK Atkinson, CA Miller and PV Hatton

School of Clinical Dentistry, University of Shefﬁeld, Shefﬁeld, UK

Introduction: Guided bone regeneration (GBR) is employed in dental implantology and the treatment of periodontal bone loss. It is based on the surgical placement of a barrier membrane that excludes soft tissue growth that would otherwise prevent bone healing [1-2].Unfortunately, non-resorbable membranes require a second operation for their removal while resorbable collagen membranes have an associated risk of disease transmission, and also existing commercial systems do not promote tissue regeneration beyond their barrier function. The aim was therefore to develop a functionalised, bilayered resorbable membrane with a dense layer to contact soft tissues, and a porous layer to promote bone healing.

Materials and methods: Compact _{films of} poly(D,L-lactide-co-glyco-lide) (PLGA, 75:25) were prepared by solvent casting from acetone. Porous membranes were prepared by dissolving PLGA (22% w/v) and nano-hydroxyapatite powder (20% w/w) [3] in acetone prior to electrospinning. Both layers were subjected to plasma polymerisation for acrylic acid grafting, and were modified by layer-by-layer (LbL) technique to generate functional polyelectrolyte (poly (styrene sulfo-nate) /poly(allyl amine) (PSS/ PAH); Sigma) to obtain 20 nanolayers to incorporate (1) an antibiotic drug (Metronidazole; Sigma) to pro-vide antibacterial activity for the compact layer, and (2) specific bone matrix peptides (KRSR and FHRRIKA; GenScript) to promote bone healing at the porous surface. The layers were assembled usingfibrin glue. Drug release from the compact layer was evaluated by UV-Vis, while in vitro biocompatibility was investigated using L292 mouse fi-broblasts (compact layer) and rat mesenchymal stromal cells (porous layer).

Results and discussion: FTIR-ATR showed that the typical absorption bands of PAH and PSS increased with layer number (i.e. SO3 stretching vibrations at 1130/cm for PSS, and NH scissoring vibra-tions at 1580/cm for PAH). The contact angle values had an alter-nate behaviour from the 9th and from 11th nanolayer for the compact and porous layer respectively. XPS spectra showed a N1s

peak at 399.5 eV and S2ppeak at 168 eV, indicating PAH and PSS

had been introduced (Fig. 1).